Odc allelic analysis method for assessing carcinogenic susceptibility

ABSTRACT

The invention includes kits and methods for assessing the susceptibility of a mammal such as a human for carcinogenesis. The methods comprise determining whether the mammal comprises a certain allele of the mammal&#39;s odc gene. The methods include use of a probe which binds specifically with a portion of one allele of the odc gene and which comprises a fluorescent label and a fluorescence quencher. The methods also include use of such a probe and a polymerase enzyme for amplifying a portion of the odc gene, the polymerase having exonuclease activity whereby the probe can be nucleolytically degraded.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/516,357 filed Mar. 1, 2000 (now U.S. Pat. No. 6,227,581). Thisapplication is entitled to priority pursuant to 35 U.S.C. §119(e) toU.S. provisional patent application No. 06/122,309, which was filed onMar. 1, 1999.

STATEMENT REGARDGING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported in part by a grant from the National Institutesof Health (NIH Grant No. R01 ES09899) and the U.S. Government maytherefore have certain rights in this invention.

REFERENCE TO A MICROFICH APPENDIX

Not applicable.

BACKGROUND OF THE INVENTION

The invention relates, in a general sense, to assessing thesusceptibility of mammals to carcinogenesis.

An individual's susceptibility to cancer is governed by the individual'sgenome and carcinogenic stimuli which the individual encounters in theenvironment. Although the carcinogenic potentials of many compounds andother stimuli (e.g., ionizing radiation) have been determined, assessingthe importance of limiting exposure to such compounds is complicated bythe fact that not all individuals are equally susceptible to thecarcinogenic effects of the compounds. Thus, the genetic component ofcarcinogenic susceptibility limits accurate prediction of cancer ratesamong individuals, even in defined environments.

Prior art observations suggest that up-regulation of the mammalian gene(odc) encoding ornithine decarboxylase is associated with enhancedsusceptibility to carcinogenesis (Luk et al., 1984, N. Engl. J. Med.311:80-83). Other prior art references indicate that overexpression ofthe odc gene increases susceptibility of mammalian cells for the tumorpromotion stage of carcinogenesis, but that over-expression of odc isnot, by itself, a sufficient condition for carcinogenesis (Clifford etal., 1995, Cancer Res. 55:1680-1686; Auvinen et al., 1992, Nature360:355-358; Moshier et al., 1993, Cancer Res. 53:2618-2622; Hibshooshet al., 1991, Oncogene 6:739-743; O'Brien et al, 1997, Cancer Res.57:2630-2637).

It is known that there are at least two alleles of the human odc gene,which have been observed as different PstI restriction fragment lengthpolymorphisms (RFLP; Hickok et al., 1987, DNA 6:179-187).

It is believed that the odc gene product, ornithine decarboxylase, isinvolved in establishing cellular polyamine levels, and that thesusceptibility of a tissue to carcinogenesis is related to polyaminelevels in the cells of the tissue. Transcription of the odc gene isactivated by the protein designated Myc (Bello-Fernandez et al., 1993,Proc. Natl. Acad. Sci. USA 90:7804-7808; Tobias et al., 1995, Oncogene11:1721-1727; Wagner et al., 1993, Cell Growth Diff. 4:789-883). Suchregulation is effected by binding of Myc/Max heterodimers to one or moreof the three Myc binding elements (also called “E-boxes”) in the humanodc gene. These E-boxes are located at about nucleotide residues −489 to−484 (relative to the transcription start site as defined by Moshier etal., 1992, Nucleic Acids Res. 20:2581-2590) in the odc 5′-promoterregion and at nucleotide residues +288 to +293 and +322 to +327 in thefirst intron of the odc gene. The fact that the E-box located in thepromoter region of the human gene is not conserved in the rat and mousegenes, suggests that the other two E-boxes are the physiologicallyimportant regulatory elements.

In normal cells, expression of c-myc (the gene encoding Myc protein) isassociated with cell cycle progression. Lack of c-myc expression permitscells to withdraw from the cell cycle and cease differentiating.Furthermore, constitutive c-myc expression promotes continuous cellcycle progression independent of growth factors (Rapp et al., 1985,Nature 317:434-438). Over-expression of c-myc is a common characteristicof many (if not most) human tumors. Prior art studies have correlatedc-myc expression and odc expression in human tumors, but not insurrounding normal (i.e., non-tumor) tissue (Gan et al., 1993, J.Histochem. Cytochem. 41:1185-1196; Mori et al., 1996, Cancer 77{8Suppl.}:1634:1638; Mimori et al., 1997, Dis. Colon Rectum 40:1095-1100).These studies suggest that deregulation of c-myc expression may lead toderegulation of odc expression.

Numerous stimuli are known which induce deregulation of c-myc expression(Schwab et al., 1984, Proc. Natl. Acad. Sci. USA 81:4940-4944; Payne etal., 1982, Nature 295:209-214; Klein et al., 1985 Immunol. Today6:208-215). Because deregulated Myc transactivates odc expression, thesestimuli can be predicted to induce deregulation of odc expression ingeneral. However, until the present invention, it has not been knownwhether either of the alleles of odc is more susceptible totransactivation by Myc, and thus the genetic component of carcinogenicsusceptibility of an individual could not be ascertained.

BRIEF SUMMARY OF THE INVENTION

The invention relates to a method of assessing the relativesusceptibility of a mammal (e.g., a human) to an epithelial cancer. Theepithelial cancer can, for example be a skin cancer (e.g., a squamouscell carcinoma), a cancer of the digestive system, an esophageal cancer,a gastric cancers, a colon cancer, a prostate cancer, a breast cancer,an hematopoietic cancer, a lung cancer, a melanoma, or a cervicalcancer. The method comprises determining whether the mammal comprises anA-allele of the odc gene. If the mammal comprises the A-allele, then themammal has a greater susceptibility to the epithelial cancer than amammal of the same type which does not comprise the A-allele.

In one embodiment, determining whether the mammal comprises an A-alleleof the odc gene comprises amplifying a reference portion of the mammal'sgenome and identifying the reference portion as corresponding to eitherthe A-allele or the G-allele. The reference portion comprises a regionof the odc gene comprising at least one nucleotide residue that ispolymorphic with respect to the A- and G-alleles. By way of example, thereference portion can be amplified using a pair of primers havingnucleotide sequences SEQ ID NOs: 3 and 4. Other primer pairs that can beused to amplify the reference portion include a primer pair having thenucleotide sequences SEQ ID NOs: 16 and 17, and a primer pair having thenucleotide sequences SEQ ID NOs: 18 and 19. Optionally, the referenceportion can be further amplified using a pair of nested primers (e.g.,primers having nucleotide sequences SEQ ID NOs: 5 and 6 if primershaving SEQ ID NOs: 3 and 4 are used for the initial amplification). Byway of example, the reference portion can comprise a region (e.g.,intron 1) of the odc gene, such as nucleotide residues +278 to +336relative to the transcription start site. The reference portion can, forexample, have a nucleotide sequence selected from the group consistingof SEQ ID NOs: 1, 2, 24, and 25). The region preferably comprises thenucleotide residue located at a position selected from the groupconsisting of −3175, −3004, −1936, +263, +317, +5294, +5915, +6697,+7487, and +7886 relative to the transcription start site, of the odcgene.

An oligonucleotide probe can be annealed with a target portion of themammal's genome prior to amplifying the reference portion. Preferably,the target portion includes a target nucleotide residue that ispolymorphic with respect to the A- and G-alleles of the odc gene. By wayof example, the target portion can include the target nucleotide residuelocated at a position selected from the group consisting of −3175,−3004, −1936, +263, +317, +5294, +5915, +6697, +7487, and +7886,relative to the transcription start site, of the odc gene. In oneembodiment, the probe is attached to a surface. The probe can,optionally, comprise a fluorescent label such as FAM, TET, rhodamine,VIC, JOE, or Hex. The probe can further comprise a fluorescence quenchersuch as TAMRA or DABCYL. For example, the probe can have a nucleotidesequence selected from the group consisting of SEQ ID NOs: 14, 20, and22, and can comprise both a fluorescent label and a fluorescentquencher. When the probe comprises both the label and the quencher, oneis preferably attached to the probe within 10 nucleotide residues of the3′-end of the probe and the other is preferably attached to the probewithin 10 nucleotide residues of the 5′-end of the probe. The referenceportion can be amplified using a DNA polymerase having 5′→3′ exonucleaseactivity, such as Thermus aquaticus DNA polymerase. Preferably, theprobe has a length from 15 to about 30 nucleotide residues and the sizeof the reference portion is not more than about 100 nucleotide residues.

In another embodiment of this method of the invention, determiningwhether the mammal comprises an A-allele of the odc gene comprisescontacting a polynucleotide derived from the mammal's genome with amolecular beacon probe. The probe has a targeting portion which iscomplementary to a target region of the odc gene, and the target regionincludes a nucleotide residue that is polymorphic with respect to the A-and G-alleles. For example, the target region can include the nucleotideresidue located at a position selected from the group consisting of−3175, −3004,−1936, +263, +317, +5294, +5915, +6697, +7487, and +7886,relative to the transcription start site, of the odc gene. Preferably,the targeting portion is completely complementary to the target regionof the A-allele of the odc gene and has a length from about 20 to about40 nucleotide residues. For example, the targeting portion of the firstprobe can have a nucleotide sequence selected from the group consistingof SEQ ID NOs: 14, 20, and 22, and the targeting portion of the secondprobe can have a nucleotide sequence selected from the group consistingof SEQ ID NOs: 15, 21, and 23. The target region can, for example,comprise about 20 to about 30 consecutive nucleotide residues of apolynucleotide having a sequence selected from the group consisting ofSEQ ID NOs: 1, 2, 24 and 25. The polynucleotide can, for example, beselected from the group consisting of a chromosome of the mammal, achromosomal fragment of the mammal, and an amplified portion of thegenome of the mammal. Optionally, the method of this embodiment canfurther comprise contacting the polynucleotide with a second molecularbeacon probe. The second probe has a targeting portion which iscompletely complementary to a target region of the G-allele of the odcgene. One or both of the probes can be attached to a discrete region ofa surface or the first probe can be attached to one surface or particleand the second probe can be attached to a different surface or particle.

The invention also relates to an isolated polynucleotide having a lengthof at least about 15 nucleotide residues and being either homologouswith or complementary to a portion of a mammalian (e.g., human) odcgene. The portion of the odc gene includes a nucleotide residue that ispolymorphic with respect to the A- and G-alleles. By way of example, thenucleotide residue can be located at a position selected from the groupconsisting of −3175, −3004, −1936, +263, +317, +5294, +5915, +6697,+7487, and +7886, relative to the transcription start site, of the odcgene. In one embodiment, the nucleotide residue at position +317 is aguanine residue, the nucleotide residue at position −3175 is a thymineresidue, and the nucleotide residue at position −3004 is a cytosineresidue. By way of example, the isolated polynucleotide can have thenucleotide sequence of one of SEQ ID NOs: 14, 15, and 20-23.

The invention further relates to a kit for assessing susceptibility of amammal to an epithelial cancer. The kit comprises a firstoligonucleotide probe which anneals specifically with a target portionof the mammal's genome and a first primer for amplifying a referenceportion of the odc gene. The target portion includes a target nucleotideresidue that is polymorphic with respect to the A- and G-alleles. Forexample, the target nucleotide residue can be the nucleotide residuelocated at a position selected from the group consisting of −3175,−3004, −1936, +263, +317, +5294, +5915, +6697, +7487, and +7886,relative to the transcription start site, of the odc gene. The firstprobe comprises a fluorescent label and a fluorescence quencher attachedto separate nucleotide residues thereof. The reference portion alsoincludes the target nucleotide residue. The kit can further comprise aDNA polymerase having 5′→3′ exonuclease activity. In one embodiment, thefirst probe is completely complementary to the target portion if thenucleotide residue located at position +317 is A, and the kit furthercomprises a second oligonucleotide probe which is completelycomplementary to the target portion if the nucleotide residue located atposition +317 is G and a second primer for performing PCR amplificationof at least the target portion in conjunction with the first primer. Forexample, the kit can comprise i) first and second oligonucleotide probeshaving nucleotide sequences SEQ ID NOs: 14 and 15, 20 and 21, or 22 and23; ii) first and second amplification primers having nucleotidesequences SEQ ID NOs: 12 and 13, 16 and 17, or 18 and 19; and iii) TaqDNA polymerase. In one embodiment, the first and second probes areattached to discrete regions of a surface or to a plurality of surfacessuch that the first probe is attached to a surface or particle discretefrom the second probe. In another embodiment, the first probe iscompletely complimentary to the target portion if the nucleotide residuelocated at position −3175 is C. This kit can further comprise a secondoligonucleotide probe which is completely complementary to the targetportion if the nucleotide residue located at position −3175 is T or asecond primer for performing PCR amplification of at least the targetportion when used with the first primer. In yet another embodiment, thefirst probe is completely complimentary to the target portion if thenucleotide residue located at position −3004 is G. This kit can furthercomprise a second oligonucleotide probe which is completelycomplementary to the target portion if the nucleotide residue located atposition −3004 is C or a second primer for performing PCR amplificationof at least the target portion when used with the first primer.

In another aspect, the invention relates to a method of assessingwhether a test compound is an inhibitor of carcinogenesis. This methodcomprises assessing ornithine decarboxylase activity in a cellcomprising an A-allele of the human odc gene in the presence of aninducer of carcinogenesis and in the presence or absence of the testcompound. If ornithine decarboxylase activity is lower in the presenceof the test compound than in the absence of the test compound, then thetest compound is an inhibitor of carcinogenesis.

The invention also relates to a method of assessing whether a testcompound is an inducer of carcinogenesis. This method comprisesassessing ornithine decarboxylase activity in a cell comprising anA-allele of the human odc gene in the presence of an inducer ofcarcinogenesis and in the presence or absence of the test compound. Ifornithine decarboxylase activity is greater in the presence of the testcompound than in the absence of the test compound, then the testcompound is an inducer of carcinogenesis.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. The invention is not limited to the precisearrangements and instrumentalities shown.

FIG. 1 is a diagram depicting an embodiment of the allelic assessmentmethod of the invention, as described herein. “R” (reporter) refers to afluorescent label (e.g., a dye such as VIC, FAM, TET, JOE, or HEX), and“Q” (quencher) refers to a fluorescence quencher (e.g., TAMRA).

FIG. 2, comprising FIGS. 2A (SEQ ID NO: 1) and 2B (SEQ ID NO: 2) are thenucleotide sequences of the A- and G-alleles, respectively, of a regionof the human odc gene extending from nucleotide residues +278 to +336,relative to the transcription start site. An asterisk marks the locationof the single nucleotide polymorphism at nucleotide residue +317 thatdifferentiates the two alleles.

FIG. 3, comprising FIGS. 3A (SEQ ID NO: 24) and 3B (SEQ ID NO: 25), arethe nucleotide sequences of the A- and G-alleles, respectively, of aregion of the human odc gene. The sequence in FIG. 3A represents aportion of the A-allele of the odc gene extending from −3363 to −2917,relative to the transcription start site, in which the nucleotideresidue at position −3175 is C and the nucleotide residue at position−3004 is G. The sequence in FIG. 3B represents a protion of the G-alleleof the odc gene extending from −3363 to −2917, relative to thetranscription start site, in which the nucleotide residue at position−3175 is T and the nucleotide residue at position −3004 is C.

FIG. 4, comprising FIGS. 4A (SEQ ID NO: 26), 4B (SEQ ID NO: 27), 4C (SEQID NO: 28), 4D (SEQ ID NO: 29), 4E (SEQ ID NO: 30), 4F (SEQ ID NO: 31),and 4G (SEQ ID NO: 32) are the nucleotide sequences of regions of theodc gene which contain single nucleotide polymorphisms. The location ofthe single nucleotide polymorphism in each sequence is indicated by anunderlined, emboldened “N”. FIG. 4A is a portion of the nucleotidesequence of the odc gene extending from −2038 to −1805, relative to thetranscription start site, surrounding the single nucleotide polymorphismat nucleotide residue −1936, wherein N is C or T. FIG. 4B is a portionof the nucleotide sequence of the odc gene extending from +149 to +382,relative to the transcription start site, surrounding the singlenucleotide polymorphism at nucleotide residue +263, wherein N is G or T.FIG. 4C is a portion of the nucleotide sequence of the odc geneextending from +5140 to +5373, relative to the transcription start site,surrounding the single nucleotide polymorphism at nucleotide residue+5294, wherein N is C or T. FIG. 4D is a portion of the nucleotidesequence of the odc gene extending from +5764 to +5997, relative to thetranscription start site, surrounding the single nucleotide polymorphismat nucleotide residue +5915, wherein N is G or A. FIG. 4E is a portionof the nucleotide sequence of the odc gene extending from +6544 to+6777, relative to the transcription start site, surrounding the singlenucleotide polymorphism at nucleotide residue +6697, wherein N can be T.FIG. 4F is a portion of the nucleotide sequence of the odc geneextending from +7402 to +7635, relative to the transcription start site,surrounding the single nucleotide polymorphism at nucleotide residue+7487, wherein N is C or T. FIG. 4G is a portion of the nucleotidesequence of the odc gene extending from +7792 to +8025, relative to thetranscription start site, surrounding the single nucleotide polymorphismat nucleotide residue +7886, wherein N can be T.

DETAILED DESCRIPTION OF THE INVENTION

It is known in the art that two alleles of the odc gene are found in thehuman population. However, the functional significance of the presenceof the two alleles was not appreciated by others. The present inventionis based on the discovery that the presence of the minor (i.e., lessfrequently occurring) allele of the odc gene (herein designated “theA-allele”) in an individual, and particularly the presence of two copiesof the minor allele in an individual, is predictive of a high incidenceof carcinogenesis in the individual. The presence of the minor allele inhuman individuals is, for example, highly predictive of the individual'ssusceptibility to development of environmentally-induced squamous cellcarcinoma and other epithelial cancers (e.g., skin cancers).

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, a mammal “comprises an A-allele of the odc gene” if thegenome of the mammal comprises one or more copies of the A-allele.

A mammal is “homozygous” for the A-allele of the odc gene if the genomeof the mammal comprises at least two copies of the A-allele.

The “A-allele” of the odc gene means a mammalian odc gene having anadenine residue at position +317 relative to the transcriptional startsite. The A-allele of the odc gene is also referred to herein as “theminor allele” of odc.

The “G-allele” of the odc gene means a mammalian odc gene having aguanine residue at position +317 relative to the transcriptional startsite. The G-allele of the odc gene is also referred to herein as “themajor allele” of odc.

A polynucleotide is “derived” from the genome of a mammal if thepolynucleotide is homologous with or complementary to at least a portionof the mammal's genome. By way of example, polynucleotides derived froma mammalian genome include, a chromosome, a chromosomal fragment, aprocessed or non-processed mRNA, a cDNA made from a processed ornon-processed mRNA, and a synthetic polynucleotide complementary to orhomologous with a portion of one of these polynucleotides.

An “isolated polynucleotide” refers to a synthetic nucleic acid segmentor a nucleic acid segment or fragment which has been separated fromsequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment, e.g., the sequences adjacent to the fragmentin a genome in which it naturally occurs. The term also applies tonucleic acids which have been substantially purified from othercomponents which naturally accompany the polynucleotide, e.g., RNA orDNA or proteins, which naturally accompany it in the cell. The termtherefore includes, for example, a recombinant DNA which is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., as a cDNA or a genomic or cDNA fragmentproduced by PCR or restriction enzyme digestion) independent of othersequences. It also includes a recombinant DNA which is part of a hybridgene encoding additional polypeptide sequence.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isanti-parallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is anti-parallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an anti-parallel fashion, at least one nucleotideresidue of the first region is capable of base pairing with a residue ofthe second region. Preferably, the first region comprises a firstportion and the second region comprises a second portion, whereby, whenthe first and second portions are arranged in an anti-parallel fashion,at least about 50%, and preferably at least about 75%, at least about90%, or at least about 95% of the nucleotide residues of the firstportion are capable of base pairing with nucleotide residues in thesecond portion. More preferably, all nucleotide residues of the firstportion are capable of base pairing with nucleotide residues in thesecond portion.

A first region of an oligonucleotide “flanks” a second region of theoligonucleotide if the two regions are adjacent one another or if thetwo regions are separated by no more than about 1000 nucleotideresidues, and preferably no more than about 100 nucleotide residues.

A second set of primers is “nested” with respect to a first pair ofprimers if, after amplifying a nucleic acid using the first pair ofprimers, each of the second pair of primers anneals with the amplifiednucleic acid, such that the amplified nucleic acid can be furtheramplified using the second pair of primers.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionfor performing a method of the invention or for associating the presenceof an A-allele of the odc gene in a mammal with carcinogenicsusceptibility. The instructional material of the kit of the inventioncan, for example, be affixed to a container which contains a kit of theinvention or be shipped together with a container which contains thekit. Alternatively, the instructional material can be shipped separatelyfrom the container with the intention that the instructional materialand the kit be used cooperatively by the recipient.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably herein.

Description

This disclosure supplements a previous disclosure (U.S. patentapplication Ser. No. 09/516,357) by the same inventors and indicatesadditional methods of assessing the presence in an individual of anA-allele of the odc gene. In this present disclosure, numericalreferences to the nucleotide residues of the odc gene have been shifted−4 in order to conform with the numbering scheme disclosed in Moshier etal. (1992, Nucleic Acids Res. 20:2581-2590). Thus, for example, theresidue designated “+321” in the previous disclosure is designated“+317” in this disclosure.

The invention includes a method of assessing the relative susceptibilityof a mammal (e.g., a human) to an epithelial cancer such as a skincancer (e.g., a squamous cell carcinoma). The ‘relative’ susceptibilityof a mammal to the epithelial cancer refers to the fact that, among apopulation of individuals exposed to equivalent carcinogenic stimuli,some individuals are more likely to develop epithelial cancers thanothers. This differential carcinogenic potential is attributable, atleast in part to the genetic makeup of the individuals in thepopulation. Germ-line mutations in tumor suppressor genes (e.g., Rb,p53, BRCA1, WT1, and the like) are one mechanism to which the geneticcontribution to carcinogenic potential may be attributed. However,germ-line mutations in tumor suppressor genes are relatively rare inlarge populations, and cannot alone account for the genetic contributionto carcinogenic potential.

Mutations in other genes, such as genes encoding proteins involved inmetabolism of carcinogens and DNA repair, have been implicated incarcinogenesis. However, even genes not involved in tumor suppression,carcinogen metabolism, or DNA repair can affect susceptibility tocarcinogenesis. For example, the gene (odc) encoding ornithinedecarboxylase may, by affecting cellular polyamine levels, affect thesusceptibility of cells to carcinogenesis, as described (Soler et al.,1998, Cancer Res. 58:1654-1659). It has not previously been knownwhether the presence in the genome of an individual of one of the twoalleles of the odc gene renders the individual more susceptible tocarcinogenesis than an individual having a genome which does notcomprise the minor allele.

The present invention is based on the discovery that the presence in thegenome of an A-allele of the odc gene is correlated with greatersusceptibility to carcinogenesis in mammals, particularly in humans. Theeffect is furthermore dose-dependent, meaning that a first individualwho is homozygous for the A-allele has greater susceptibility than asecond individual who is heterozygous for the A- and G-alleles, and thatthe second individual has greater susceptibility than a third individualwho is homozygous for the G-allele. Thus, the method of the inventionfor assessing relative susceptibility of a mammal to an epithelialcancer comprises determining whether the mammal comprises an A-allele ofthe odc gene. If the mammal comprises the A-allele, then the mammal hasgreater susceptibility to the epithelial cancer than a mammal of thesame type which does not comprise the A-allele. The invention preferablycomprises determining whether the mammal is homozygous for the A-allele,heterozygous for the A- and G-alleles, or homozygous for the G-allelebecause, as noted above, the carcinogenic susceptibility attributable tothese genotypes differs.

Substantially any method of detecting an allele of the odc gene can beused including, for example, hybridization, amplification, or sequencingmethods. For example, a PCR/RFLP method can be used to determine whetherthe mammal is homozygous for the A-allele, heterozygous for the A- andG-alleles, or homozygous for the G-allele. This PCR/RFLP method relieson the fact that the A-allele of the odc gene comprises a PstIrestriction endonuclease cleavage site at nucleotide residues +313 to+318, relative to the transcription start site, while the G-allele doesnot. The size of restriction fragments generated by treating apolynucleotide comprising residues +313 to +318 of the odc gene withrestriction endonuclease PstI thus depends on whether the polynucleotideis derived from an A-allele or a G-allele of the gene. If thepolynucleotide is derived from an A-allele, there will be one morerestriction fragment following reaction with PstI than if it is derivedfrom a G-allele.

The PCR/RFLP method of the invention thus comprises amplifying a portionof the odc gene (comprising nucleotide residues +313 to +318) of amammal, treating the amplified portion with PstI, and observing at leastone of the size and the number of restriction fragments so generated.Acceptable buffers and reaction conditions for amplification andrestriction endonucleolytic digestion are known in the art. By way ofexample, if the odc gene of a human individual is amplified using anested pair of primers (e.g., using a first pair of primers havingsequences SEQ ID NOs: 3 and 4 and then a second pair of primers havingsequences SEQ ID NOs: 5 and 6), a 546-nucleotide-residue portion isamplified. Treatment of this amplified portion with PstI yields a(non-cleaved) 546-nucleotide-residue polynucleotide if the portion wasderived from a G-allele and both 351- and 195-nucleotide-residuepolynucleotides if the portion was derived from an A-allele. If bothalleles from the individual are amplified and treated together, only a546-nucleotide-residue polynucleotide will be observed if the individualis homozygous for the G-allele, a mixture (ca. 1:1) of 351- and195-nucleotide-residue polynucleotides will be observed if theindividual is homozygous for the A-allele, and a mixture (ca. 1:1:1) of351-, 195-, and 546-nucleotide-residue polynucleotides will be observedif the individual is heterozygous for the A- and G-alleles.

Another method of determining whether the individual mammal comprises anA-allele of the odc gene comprises amplifying and sequencing a referenceportion of the mammal's genome. The reference portion includes at leasta region of the odc gene, preferably including both alleles from theindividual. The region includes at least one nucleotide residue that ispolymorphic with respect to the A- and G-alleles. For example, each ofthe nucleotide residues at positions −3175, −3004, −1936, +263, +317,+5294, +5915, +6697, +7487, and +7886, relative to the transcriptionstart site, of the odc gene, differs between the A- and G-alleles. Theresidue at position +317 is adenine in the A-allele and guanine in theG-allele. The residue at position −3175 is cytosine in the A-allele andthymine in the G-allele. The residue at position −3004 is guanine in theA-allele and cytosine in the G-allele. Examples of other polymorphicresidues are described in FIG. 4. Other polymorphic sequences whichdiffer between the A- and G-alleles of the odc gene can be used toassess which allele is present in a human, and the invention is notlimited to those disclosed herein.

The reference portion of the odc gene can be amplified, for example,using standard PCR primer pairs which anneal with complementary strandsat positions flanking the region. For example, primer pairs which can beused to amplify the reference portion include the oligonucleotideshaving the sequences SEQ ID NOs: 12 and 13, 16 and 17, and 18 and 19.Alternatively, nested pairs of primers can be used to specificallyamplify the region of the odc gene. By way of example, a region of thehuman odc gene can be amplified using a first pair of primers having thenucleotide sequences SEQ ID NOs: 3 and 4, using a second pair of primershaving the nucleotide sequences SEQ ID NOs: 5 and 6, or using both thefirst and second pairs of primers.

For example, when the human odc gene is amplified using the second pairof primers having the nucleotide sequences SEQ ID NOs: 5 and 6 (alone orin conjunction with the first pair of primers having the nucleotidesequences SEQ ID NOs: 3 and 4), the amplified region will comprise thenucleotide sequence SEQ ID NO: 1 if the gene is the A-allele or thenucleotide sequence SEQ ID NO: 2 if the gene is the G-allele. When bothalleles from an individual are amplified together, the amplified regionswill comprise the nucleotide sequence SEQ ID NO: 1, but not SEQ ID NO:2, if the individual is homozygous for the A-allele. The amplifiedregions will comprise the nucleotide sequence SEQ ID NO: 2, but not SEQID NO: 1, if the individual is homozygous for the G-allele. Theamplified regions will comprise a mixture of polynucleotides, somehaving the nucleotide sequence SEQ ID NO: 1 and other having thenucleotide sequence SEQ ID NO: 2 if the individual is heterozygous forthe A- and G-alleles. The odc genotype of the individual can thereby bedetermined. SEQ ID NOs: 1 and 2 are listed in FIGS. 2A and 2B,respectively.

A requirement that amplified genomic regions of individuals be sequencedcan be detrimental to the efficiency and practicality of a large-scale(i.e., “high-throughput”) screening effort. Thus, if the odc genotypesof large numbers (i.e., more than about 50 to 500) of individuals are tobe determined, it is preferable to use a screening method that does notrequire sequencing of individual amplified nucleic acids. An example ofone such method is now described.

The invention includes an allelic discrimination method for identifyingthe odc genotype of an individual mammal (e.g., a human). A summary ofthis method is depicted in FIG. 1. The allelic discrimination method ofthe invention involves use of a first oligonucleotide probe whichanneals with a target portion of the mammal's genome. The target portioncomprises a portion of the odc gene of the mammal, including a targetnucleotide residue that is polymorphic with respect to the A- andG-alleles. For example, the target nucleotide residue can be located ata position selected from the group consisting of −3175, −3004, −1936,+263, +317, +5294, +5915, +6697, +7487, and +7886, relative to thetranscription start site. Because the nucleotide residue at any one ofthese positions differs in the A-allele and the G-allele, the firstprobe is completely complementary to only one of the two alleles.Alternatively, a second oligonucleotide probe can also be used which iscompletely complementary to the target portion of the other of the twoalleles. The allelic discrimination method of the invention alsoinvolves use of at least one, and preferably a pair of amplificationprimers for amplifying a reference region of the odc gene of the mammal.The reference region includes at least a portion of the target portion,and preferably includes the target nucleotide residue.

The allelic discrimination method of the invention is performed byannealing the first probe with the target portion of an odc geneobtained from an individual mammal and amplifying the reference regionof the odc gene, which comprises at least part of the target portion.Alternatively, both the first and the second probe can be annealed withthe target portion in order to achieve completely complementaryannealing of a probe to each target portion.

Because the reference region and the target portion overlap by at leastone base, preferably by at least about half the length of the targetportion, and more preferably completely overlap, the enzyme (e.g.,Thermus aquaticus {Taq} DNA polymerase) which catalyzes theamplification reaction and the first (or second) probe will collide. Ifthe probe is not completely complementary to the target portion, it ismore likely to dissociate from the target portion upon collision than ifit is completely complementary. Therefore, unless the enzyme exhibits5′→3′ exonuclease activity, amplification ceases or is greatlyinhibited.

If the enzyme which catalyzes the amplification reaction exhibits 5′→3′exonuclease activity (e.g., Taq DNA polymerase), then the enzyme will atleast partially degrade the 5′-end of a probe with which it collidesunless the probe dissociates from the target portion upon collision withthe enzyme. As noted above, if the probe is not completely complementaryto the target portion, it is much more likely to dissociate from thetarget portion upon collision than if it is completely complementary. Ifa detectable label is attached to a nucleotide residue at or near the5′-end of the probe, release of the detectable label from the probe canbe used as an indication that the enzyme and probe have collided andthat the probe did not dissociate from the target portion. Thus, releaseof the detectable label from the probe upon amplification of the regionindicates that the probe was completely complementary to the targetportion. By selecting either or both of a probe completely complementaryto the target portion of the A-allele of the odc gene and a probecompletely complementary to the target portion of the G-allele of thegene and assessing release of the label from the probe(s), the identityof the allele(s) can be ascertained.

The probe is preferably a DNA oligonucleotide having a length in therange from about 20 to about 40 nucleotide residues, preferably fromabout 20 to about 30 nucleotide residues, and more preferably having alength of about 25 nucleotide residues. In one embodiment, the probe isrendered incapable of extension by a PCR-catalyzing enzyme such as Taqpolymerase, for example by having a fluorescent probe attached at one orboth ends thereof. Although non-labeled oligonucleotide probes can beused in the kits and methods of the invention, the probes are preferablydetectably labeled. Exemplary labels include radionuclides,light-absorbing chemical moieties (e.g., dyes), fluorescent moieties,and the like. Preferably, the label is a fluorescent moiety, such as6-carboxyfluorescein (FAM), 6-carboxy-4,7,2′, 7′-tetrachlorofluoroscein(TET), rhodamine, JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein),or HEX (hexachloro-6-carboxyfluorescein).

In a particularly preferred embodiment, the probe of the inventioncomprises both a fluorescent label and a fluorescence-quenching moietysuch as 6-carboxy-N,N,N′,N′-tetramethylrhodamine (TAMRA, or4-(4′-dimethlyaminophenylazo)benzoic acid (DABCYL). When the fluorescentlabel and the fluorescence-quenching moiety are attached to the sameoligonucleotide and separated by no more than about 40 nucleotideresidues, and preferably by no more than about 30 nucleotide residues,the fluorescent intensity of the fluorescent label is diminished. Whenone or both of the fluorescent label and the fluorescence-quenchingmoiety are separated from the oligonucleotide, the intensity of thefluorescent label is no longer diminished. Preferably, the probe of theinvention has a fluorescent label attached at or near (i.e., withinabout 10 nucleotide residues of) one end of the probe and afluorescence-quenching moiety attached at or near the other end.Degradation of the probe by a PCR-catalyzing enzyme releases at leastone of the fluorescent label and the fluorescence-quenching moiety fromthe probe, thereby discontinuing fluorescence quenching and increasingthe detectable intensity of the fluorescent label. Thus, cleavage of theprobe (which, as discussed above, is correlated with completecomplementarity of the probe with the target portion) can be detected asan increase in fluorescence of the assay mixture.

If detectably different labels are used, more than one labeled probe canbe used. For example, the assay mixture can contain a first probe whichis completely complementary to the target portion of the A-allele of theodc gene and to which a first label is attached, and a second probewhich is completely complementary to the target portion of the G-allele,as described herein in Example 2. When two probes are used, the probesare detectably different from each other, having, for example,detectably different size, absorbance, excitation, or emission spectra,radiative emission properties, or the like. For example, a first probecan be completely complementary to the target portion of the A-alleleand have FAM and TAMRA attached at or near opposite ends thereof. Thefirst probe can be used in the method of the invention together with asecond probe which is completely complementary to the target portion ofthe G-allele and has TET and TAMRA attached at or near opposite endsthereof. Fluorescent enhancement of FAM (i.e., effected by cessation offluorescence quenching upon degradation of the first probe by Taqpolymerase) can be detected at one wavelength (e.g., 518 nanometers),and fluorescent enhancement of TET (i.e., effected by cessation offluorescence quenching upon degradation of the second probe by Taqpolymerase) can be detected at a different wavelength (e.g., 582nanometers).

It is important that the probe exhibit a melting temperature (Tm) withinthe range from about 60 to 70° C., more preferably in the range from 65to 67° C.

Furthermore, because each probe is completely complementary to only oneof the A- and G-alleles of the odc gene, each probe will necessarilyhave at least one nucleotide residue which is not complementary to thecorresponding residue of the other allele. This non-complementarynucleotide residue of the probe is preferably located near themidsection of the probe (i.e., within about the central third of theprobe sequence) and is preferably approximately equidistant from theends of the probe. Thus, for example, the probe which is completelycomplementary to the A-allele of the human odc gene can, for example, becompletely complementary to nucleotide residues +306 to +325 of theA-allele, relative to the transcription start site. Because the A- andG-alleles differ at position +317, this probe will have a mismatchedbase pair nine nucleotide residues from one end when it is annealed withthe corresponding target portion of the G-allele.

Essentially any probe corresponding to a polymorphic portion of the odcgene can be used, so long as it has a nucleotide sequence whichcorresponds to at least one nucleotide that is polymorphic with respectto the A- and G-alleles (i.e., at least one nucleotide residue is notidentical between the A-allele and the G-allele). By way of example,labeled probes having the sequences SEQ ID NOs: 14 and 15 can be used,in conjunction with amplification primers having sequences SEQ ID NOs:12 and 13, in order to determine the allelic content of a mammal (i.e.,to assess whether the mammal comprises one or both of an A-allele and aG-allele of the odc gene). As another example, labeled probes having thesequences SEQ ID NOs: 20 and 21 can be used, in conjunction withamplification primers having sequences SEQ ID NO: 16 and 17, in order todetermine the allelic content of a mammal. Alternatively, labeled probeshaving the sequences SEQ ID NOs: 22 and 23 can be used, in conjunctionwith amplification primers having sequences SEQ ID NOs: 18 and 19, inorder to determine the allelic content of a mammal.

The size of the reference portion which is amplified according to theallelic discrimination method of the invention is preferably not morethan about 100 nucleotide residues. In one embodiment, the Tm for theamplified reference portion with the genomic DNA or fragment thereof isin the range from about 57 to 61° C.

It is understood that binding of the probe(s) and primers and thatamplification of the reference portion of the odc gene according to theallelic discrimination method of the invention will be affected by,among other factors, the concentration of Mg⁺⁺in the assay mixture, theannealing and extension temperatures, and the amplification cycle times.Optimization of these factors requires merely routine experimentationand is within the ken of the skilled artisan.

The invention further includes another allelic discrimination method.The probes used in this method are generally known as “molecularbeacons.” Molecular beacon probes are single-stranded oligonucleotideshaving a fluorescent label (e.g., rhodamine, FAM, TET, VIC, JOE, or HEX)attached to the 5′-end thereof and a fluorescence quencher (e.g., TAMRAor DABCYL) attached to the 3′-end thereof (or vice versa), as described(Kostrikis et al., 1998, Science 279:1228-1229). The sequence of eachmolecular beacon probe is selected to include two complementary hairpinregions, whereby the probe can self-anneal to form a hairpin structure.The 5′-and 3′- ends are brought into close association when the hairpinstructure forms. The probe also comprises a targeting portion which isselected to be complementary to a target sequence (e.g., a single alleleof a gene). The targeting portion and at least one of the hairpinregions are located in close proximity to one another, meaning that thetargeting portion either overlaps the hairpin region or flanks it,having no more than about 5 nucleotide residues therebetween.

If the hairpin regions of the molecular beacon probe anneal with oneanother, then the probe does not fluoresce, because the hairpinstructure forms and the fluorescence quencher attached to one end of theprobe quenches fluorescence of the label attached to the other end ofthe probe. If the targeting portion of the probe anneals with a regionof a nucleic acid having the target sequence, then formation of thehairpin structure is inhibited, the fluorescence quencher is not broughtinto association with the fluorescent label, and the probe fluoresces.Multiple molecular beacon probes can be used in a single reactionmixture, and fluorescence associated with the probes can bedifferentiated if, for example, the labels attached to the probes aredetectably different (e.g., fluorescent emissions from the dyes havedifferent wavelengths). For example, using a first molecular beaconprobe which anneals specifically with one allele of a gene (e.g., odc)and a second, differently labeled molecular beacon probe which annealsspecifically with the other allele, it is possible to differentiateindividuals homozygous for the one allele, individuals homozygous forthe other allele, and individuals heterozygous for the two alleles.

Thus, according to this method of the invention, a molecular beaconprobe is used which has a targeting portion which is complementary to atarget region (e.g., 20 to 40 nucleotide residues, more preferably 20 to30 residues) of the odc gene, the target region including, andpreferably being approximately centered around a target nucleotideresidue that is polymorphic with respect to the A- and G-alleles. Forexample, the target nucleotide residue can be located at a positionselected from the group consisting of −3175, −3004, −1936, +263, +317,+5294, +5915, +6697, +7487, and +7886, relative to the transcriptionstart site. More preferably, two such probes are used, one having atargeting region completely complementary to the target region of theA-allele of the odc gene, and the other having a targeting regioncompletely complementary to the target region of the G-allele.

The method of determining whether the mammal comprises an A-allele ofthe odc gene using a molecular beacon probe comprises contacting apolynucleotide derived from the mammal's genome with the probe. Theprobe has a targeting region which is complementary (preferablycompletely complementary) to a target region of one allele the mammal'sodc gene. The target region includes a target nucleotide residue that ispolymorphic with respect to the A- and G-alleles. For example, thetarget nucleotide residue can be located at a position selected from thegroup consisting of −3175, −3004, −1936, +263, +317, +5294, +5915,+6697, +7487, and +7886, relative to the transcription start site. Ifthe targeting region is complementary to the target region of at leastone allele of the odc gene of the mammal, then the probe fluoresces;otherwise, it does not fluoresce. Two such probes can be used (e.g., onecompletely complementary to the target region of the A-allele and theother completely complementary to the target region of the G-allele),and if the two probes are detectably different (e.g., they havefluorescent labels which fluoresce at different wavelengths) then thepresence of two alleles can be simultaneously assayed.

Of course, it is understood that any method of ascertaining the genotypeof an individual mammal at the odc locus can be used to assess therelative susceptibility of the individual to an epithelial cancer. Thus,the invention includes known methods (both those described herein andthose not explicitly described herein) and allelic discriminationmethods which may be hereafter developed.

The invention also includes a kit for assessing the susceptibility of amammal to an epithelial cancer according to the one or more of themethods of the invention. The kit comprises a plurality of reagentsuseful for performing one of the methods (e.g., one or more of the probeor primer pairs described herein, and optionally further comprises aninstructional material which describes how the method is performed, theassociation between the presence of the A-allele and carcinogenicsusceptibility, or both. The instructional material of the kit of theinvention can, for example, be affixed to a container which contains oneor more reagents used in a method of the invention or be shippedtogether with a container which contains the reagent(s). Alternatively,the instructional material can be shipped separately from the containerwith the intention that the instructional material and the reagent(s) beused cooperatively by the recipient.

By way of example, a kit for performing the allelic discriminationmethod of the invention comprises:

a) a first oligonucleotide probe which anneals specifically with targetportion of the mammal's genome, wherein the target portion includes atarget nucleotide residue located at a position selected from the groupconsisting of −3175, −3004, −1936, +263, +317, +5294, +5915, +6697,+7487, and +7886, relative to the transcription start site, of the odcgene, the probe comprising a fluorescent label and a fluorescencequencher attached to separate nucleotide residues thereof, and

b) a primer for amplifying a reference portion of the odc gene, thereference portion including the target nucleotide residue.

The kit can further comprise a DNA polymerase having 5′→3′ exonucleaseactivity. The kit can also comprise a second oligonucleotide probehaving a different annealing specificity than the first (e.g., whereinthe first is completely complementary to the target portion of the Aallele and the second is completely complementary to the target portionof the G allele), a second primer (e.g., such that this and the firstprimer can be used to amplify at least the target portion by a PCR), orboth. The kit can comprise an instructional material which can, forexample, describe performance of the allelic discrimination method, theassociation between the presence of the A-allele and carcinogenicsusceptibility, or both.

The invention includes another kit, this kit comprising at least one,and preferably two molecular beacon probes, as described herein. Whenthe kit comprises two molecular beacon probes, one is preferablyspecific for (i.e., completely complementary to a region including thetarget nucleotide residue, such as one of residues +317, −3004, and−3175) the A-allele of the odc gene, and the other is specific for theG-allele. This kit can further comprise an instructional material, asdescribed above.

In certain embodiments, the oligonucleotide probes used in the kits andmethods described herein can be attached to a surface in order tofacilitate handling of the oligonucleotide probes. Each of theoligonucleotide probes can be linked with a plurality of surfaces (e.g.,oligonucleotide probes for a particular nucleotide polymorphism beingattached to a particle discrete from a particle to which oligonucleotideprobes for another nucleotide polymorphism are attached), or they can beattached to discrete regions of a single surface (e.g., as in theGENECHIP™ device of Affymetrix, Inc.). Annealing between individualoligonucleotide probes and the polymorphism corresponding thereto can bedetected using standard methods.

The invention also includes a screening method for assessing whether atest compound is an inhibitor of carcinogenesis. Because it has beendiscovered, as described herein, that it is the A-allele of the odc genewhich is more strongly correlated with carcinogenic susceptibility,discovery of agents which inhibit expression of this allele specifically(i.e., without inhibiting expression of the G-allele as severely) isuseful for development of carcinogenesis-inhibiting pharmaceutical andother compositions. This screening method comprises assessing ornithinedecarboxylase activity in a cell which comprises an A-allele of thehuman odc gene. The cell is preferably homozygous for the A-allele.Ornithine decarboxylase activity is assessed in the presence of aninducer of carcinogenesis and in the presence or absence of the testcompound. If ornithine decarboxylase activity is lower in the presenceof the test compound than in the absence of the test compound, then thetest compound is an inhibitor of carcinogenesis. If ornithinedecarboxylase activity is greater in the presence of the test compoundthan in the absence of the test compound, then the test compound is aninducer of carcinogenesis. This screening method can optionally includea step wherein a cell or cell line comprising an A-allele of the odcgene is selected prior to contacting the cell with the test compound andassessing ornithine decarboxylase activity of the cell. Optionally, acell which is homozygous for the G-allele of odc can be treatedidentically, and ornithine decarboxylase activity of this cell assessedas a control. If the test compound has a different effect on ornithinedecarboxylase activity in the cell comprising the A-allele than on thesame activity in the cell comprising the G-allele, then this is anindication that the effects of the test compound on mammalian cells willdepend on the genotype of the mammalian cells.

Although the descriptions provided herein are principally directed tokits and methods which are applicable to human cancers, it will beunderstood by the skilled artisan that such methods and kits aregenerally applicable to cancers of mammals of all sorts. Modification,where necessary, of the kits and methods of the invention to conform tonon-human cancers is understood, and the ordinarily skilled veterinaryworker can design and perform such modification with merely ordinary, ifany, experimentation. Representative mammals include, for example,primates, cattle, pigs, horses, sheep, cats, and dogs.

The kits and methods described herein are applicable for substantiallyany epithelial cancer, including, for example, skin cancers such assquamous cell carcinoma. Other epithelial cancers for which thecompositions, kits, and methods described herein can be used includecancers of the digestive system, esophageal cancers, gastric cancers,colon cancers, prostate cancers, breast cancers, hematopoietic cancers,lung cancers, melanomas, and cervical cancers.

It is believed that the nucleotide sequence of the G-allele of the humanodc gene has not been previously described. The invention thereforeincludes an isolated polynucleotide which is homologous with orcomplementary to the G-allele of the odc gene, wherein thepolynucleotide is homologous with or complementary to, respectively, anucleotide residue at position +317, −3004, or −3175, relative to thetranscription start site. Such polynucleotides include, for example,cDNA generated from a mammalian (e.g., human) transcript (e.g., aprimary transcript or an mRNA) of the odc gene or a probe which iscomplementary to or homologous with a portion of the odc gene includingnucleotide residue +317. Examples of such a polynucleotide include apolynucleotide having the nucleotide sequence SEQ ID NO: 2, apolynucleotide having the nucleotide sequence SEQ ID NO: 10, or apolynucleotide having the nucleotide sequence SEQ ID NO: 11. Thepolynucleotide has a length of at least about 15 nucleotide residues,and is preferably in the range from about 20 to 100 residues, from 20 to40 residues, or more preferably, 20 to 30 (e.g., 25) nucleotideresidues. The invention also includes analogous isolated polynucleotidescorresponding to the A-allele of the odc gene.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teaching providedherein.

EXAMPLE 1

Others (Hickok et al., 1987, DNA 6:179-187) have observed thatapproximately 50% of human individuals are homozygous for the majorallele of the odc gene (herein designated the G-allele) andapproximately the remainder are heterozygous for the G-allele and theminor allele of the odc gene, herein the A-allele. A nested PCR/RFLPassay was used to confirm this analysis in 51 human individuals. Theresults of this assay are listed in Table 1.

TABLE 1 Number of Individuals Genotype¹ % of All Individuals 33 G G 65.716 A G 31.4 2 A A 3.9 Notes: ¹G means a G-allele of the odc gene; Ameans an A-allele of the odc gene.

The PCR/RFLP assay was performed as follows. Genomic DNA was obtainedfrom individuals. A 756-nucleotide-residue fragment containing the PstIsite at +313 to +318 (relative to the transcription start site) of theodc gene was amplified using a first pair of primers, and then amplifiedusing a second pair of (nested) primers to yield a546-nucleotide-residue amplified fragment. The nucleotide sequences ofthe first pair of primers were:

5′-ATCGTGGCTG GTTTGAGCTG-3′ (SEQ ID NO: 3) and

5′-GTCATCTGCT CTGTAGACAC AGCG-3′ (SEQ ID NO: 4). Amplification using thefirst pair of primers yielded a fragment having a length of about 756nucleotide residues. The nucleotide sequences of the second pair ofprimers were:

5′-GGTGCTATAA GTAGGGAGCG CC-3′ (SEQ ID NO: 5) and

5′-CCGAAGGGTT GGGAAAGAGG-3′ (SEQ ID NO: 6). Amplification using thesecond pair of primers yielded a fragment having a length of about 546nucleotide residues. This amplified fragment was purified from anagarose gel and digested using PstI. The digested amplified fragmentpreparation was loaded onto an agarose gel and electrophoresed.Amplified fragments obtained from individuals homozygous for theG-allele were not cut by PstI, and exhibited a relative mobilitycorresponding to the uncut 546-nucleotide-residue fragment. Amplifiedfragments obtained from individuals homozygous for the A-alleleexhibited relative mobilities corresponding to fragments of 351 and 195nucleotide residues. Amplified fragments obtained from heterozygousindividuals exhibited relative mobilities corresponding to fragments of546, 351, and 195 nucleotide residues.

The portion of the human odc gene extending from nucleotide residues+278 to +336 (relative to the transcription start site) was sequenced inindividuals homozygous for either the A-allele or the G-allele. Thenucleotide sequences of this region for the A- and G-alleles areindicated in FIGS. 2A (SEQ ID NO: 1) and 2B (SEQ ID NO: 2),respectively. The failure of PstI to cleave the G-allele is attributableto the presence of a guanine residue at nucleotide residue +317.

Without wishing to be bound by any particular theory of operation, it isbelieved that the following discussion explains the increasedcarcinogenic susceptibility associated with the presence in a mammaliangenome of the A-allele of the odc gene, relative to the presence of theG-allele. The single nucleotide polymorphism (SNP) at nucleotide residue+317 is in a very important region of the odc gene. The SNP is withinintron 1, close to exon 1. This region contains two Myc-binding domains,designated MB1 and MB2 (Bello-Fernandez et al., 1993, Proc. Natl. Acad.Sci. USA 90:7804-7808). These Myc-binding domains are responsible forthe known ability of Myc to transactivate odc effected by binding ofMyc/Max heterodimers to these domains. However, binding of Max/Maxhomodimers does not appear to transactivate odc transcription. Thus, therelative intracellular amounts of Myc, Max, and possibly Mad proteinsdetermine the extent of odc transcription. Clearly, however, Myc/Maxheterodimer formation is favored over Max/Max homodimerization(Blackwood et al., 1992, Genes Dev. 6:71-80). It follows that wheneverc-myc expression is enhanced, odc expression will also be enhanced.

It is furthermore known that the sequence context surrounding MB1 andMB2 differentially affects relative binding of Myc/Max and Max/Maxdimers (Solomon et al., 1993, Nucl. Acids Res. 21:5372-5376). Binding ofMyc/Max dimers is much more sensitive to the nature of the flankingsequences than Max/Max dimers. Bases located −1 to −3, relative to thehexanucleotide core of the binding site (CACGTG; SEQ ID NO: 7) are knownto be particularly important for binding, but sequence context furtherfrom the core has not been extensively investigated. Recently, the baseat position −4 relative to the hexanucleotide core has been shown toinfluence binding of Myc/Max dimers (Walhout et al., 1998, Biochim.Biophys. Acta 1397:189-201). The SNP in the human odc gene is atposition −5, relative to the MB2 domain, and the adenine residue at thisposition is only found in this human allele. In the rat and mouse genes,there is a guanine residue at this position. Despite the fact that thereexists an odc allele with an adenine residue at position +317 (becausesome humans have a PstI restriction site beginning at base +313), allreported functional analyses of the three human Myc-binding elements(i.e., including the site in the 5′upstream promoter region and the twosites in intron 1) appear to have been made using genes having a guanineresidue at nucleotide residue +317. There have been no functionalcomparisons of the A-allele of the human gene and the G-allele withrespect to regulation by c-myc.

In order to determine if the SNP located at nucleotide residue +317 inthe human odc gene has functional significance, preliminary gel shiftanalyses were performed using oligonucleotide probes specific for the A-and G-alleles. In separate experiments, the promoter strength of the A-and G-alleles of human odc (i.e., the ability of these alleles to inducetranscriptions of operably linked cDNA encoding luciferase enzyme) wereassessed in transfection assays.

Gel shift analyses were performed as follows. Complexes of HeLa cellnuclear proteins and ³²P-labeled oligonucleotide probes were formed byincubating the proteins and probes together at room temperature for 30minutes. The probes used to detect the A-allele had the nucleotidesequences:

5′-CCGGCCTGCA GAGACACGTG GTCGCCGAGC G-3′ (SEQ ID NO: 8) and

5′-GGCCGGACGT CTCTGTGCAC CAGCGGCTCG C-3′ (SEQ ID NO: 9). The probes usedto detect the G-allele had the nucleotide sequences:

5′-CCGGCCTGCG GAGACACGTG GTCGCCGAGC G-3′ (SEQ ID NO: 10) and

5′-GGCCGGACGC CTCTGTGCAC CAGCGGCTCG C-3′ (SEQ ID NO: 11). Thesenucleotide sequences correspond to odc nucleotide residues +301 to +332,relative to the transcription start site, and had either an adenineresidue (corresponding to the A-allele) or a guanine residue(corresponding to the G-allele) at position +317. Oligonucleotides wereend-labeled using T4 polynucleotide kinase and gamma-³²P-ATP.

The electrophoretic mobility shift assays were performed as follows. Theassays were performed in 10 microliter reaction mixtures which consistedof 50 millimolar Tris—HCl at pH 7.5, 5 millimolar MgCl₂, 2.5 millimolarEDTA, 2.5 millimolar dithiothreitol, 250 millimolar NaCl, 0.25micrograms per microliter poly-dI:dC, 20% (v/v) glycerol, 5×10⁵ to10×10⁵ counts per minute of ³²P-end labeled probes, and an aliquot ofHeLa cell nuclear extract containing about 10 micrograms of protein.

The resulting protein-probe complexes were electrophoresed on anon-denaturing 4% (w/v) polyacrylamide gel comprising 0.5× TBE buffer.The electrophoresis procedure indicated that the fragment of theG-allele formed a faster-migrating complex with nuclear proteins thanthe fragment of the A-allele. Furthermore, there was significantly morebinding of nuclear proteins to the probe corresponding to the A-allele.These data demonstrate that the A- and G-alleles do not have identicalbinding properties with respect to nuclear proteins, suggesting that thetwo alleles are differentially regulated.

In order to study the functional differences between the A- andG-alleles of odc in vivo, reporter constructs were made. The fragmentcorresponding to about nucleotide residues −466 to +3066 of the humanodc gene (relative to the transcription start site) was isolated by PCRfrom individuals who were homozygous for either the A- or G-allele. Theisolated fragments were cloned into the pGL3 plasmid (Promega Corp.,Madison, Wis.) and operably linked with a modified firefly luciferasegene. Using a standard calcium phosphate method, NIH 3T3 cells weretransient transfected using 1 microgram these constructs together with0.1 microgram of a renilla luciferase plasmid (pRL; Promega Corp.,Madison, Wis.). Both luciferase activities were assayed in cell extractharvested 24 hours post-transfection and in cells transfected with emptypGL3 plasmid as a control.

The results of these assays are listed in Table 2.

TABLE 2 Luciferase Activity¹ Experiment # A-Allele G-Allele A/G AlleleRatio 1 39.7 13.3 2.98 2 38.9 12.1 3.21 3 28.4 8.6 3.30 4 22.6 8.9 2.545 22.9 6.8 3.37 Summary 3.08 ± 0.3 (n = 5) Notes: ¹Luciferase activityis expressed as a ratio of firefly luminescence (units per microgram) torenilla luminescence (units per microgram) measured using the sameextract.

The magnitude of luciferase activity expressed in transfected cellsreflected the relative promoter strength of the promoters of the A- andG-alleles. The results indicated the promoter/intron 1 region of theA-allele yielded consistently greater luciferase expression than did thesame region of the G-allele. Presumably, most, if not all, of the odeallele-driven reporter gene expression in these experiments is due toendogenous Myc and Max present in the nuclei of the transfected cells.

EXAMPLE 2

Protocol for ABIPRISM™ 7700 Allelic

Discrimination of ODC Promoter Region

In this Example, a specific example of a protocol useful for determiningthe allelic composition of a mammal is provided. This protocol isespecially adapted for use with the ABI PRISM™ 7700 Sequence DetectionSystem (PE Biosystems, Foster City, Calif.) and a TAQMAN™ (RocheMolecular Systems, Inc., Branchburg, N.J.) PCR reagent kit. In thisprotocol, the following oligonucleotide primers and probes are used, theoligonucleotides having the indicated melting temperatures (Tm).

“Forward primer”: 5′-CCT GGG CGC TCT GAG GT-3′ (Tm = 58.4° C.; SEQ IDNO: 12) “Reverse primer”: 5′-AGG AAG CGG CGC CTC AA-3′ (Tm = 61.5° C.;SEQ ID NO: 13) “TAQMAN™ Allele A Probe”: 5′-VIC-CAC GTG TCT CTG CAG GCCGG-TAMRA-3′ (Tm = 66° C.; SEQ ID NO: 14; VIC is a proprietaryfluorescent dye available from PE Biosystems; TAMRA is6-carboxytetramethylrhodamine)) “TAQMAN™ TM Allele G Probe”: 5′-FAM-CACGTG TCT CCG CAG GCC G-TAMRA-3′ (Tm = 67° C.; SEQ ID NO: 15; FAM is6-carboxyfluorescein)

The ABI PRISM™ system detects fluorescence which arises from release ofa fluorescent dye from a TAQMAN™ probe during PCR. For either probe, VICor FAM can be replaced with JOE(2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), with TET(tetrachloro-6-carboxyfluorescein), or with HEX(hexachloro-6-carboxyfluorescein). Each of VIC, FAM, JOE, and HEX isquenched by TAMRA when attached to different residues of anoligonucleotide (e.g., when the dye is attached at one end and TAMRA isattached at the other end).

The protocol is as follows

1) Prepare 5.39 milliliters of a “PCR Master Mix”, as indicated in thefollowing table.

Stock microliters microliters/ Final Concen- per reaction 110 reactionconcen- Reagent tration mixture mixtures tration TAQMAN ™ 2x 25 2,750 1xUniversal PCR Master Mix (PE Biosystems part number 4304437) ForwardPrimer 10 3 330 600 nanomolar Reverse Primer 10 3 330 600 nanomolarTAQMAN ™ 100 1.25 of a 137.5 250 Allele A Probe 1/10 dilution nanomolarTAQMAN Allele G 100 1.25 of a 137.5 250 Probe 1/10 dilution nanomolarDistilled, deionized — 15.5 1,705 — water Total Volume — 49 5,390 1x

2) Load 49 microliters of the PCR Master Mix into each well of anABIPRISM™ 96-well optical reaction plate (PE Biosystems part numberN801-0560).

3) Add 100 nanograms (in 1 microliter volume) of each of three DNAstandards (homozygous AA, homozygous GG, and no template controls)individually to eight wells each (i.e., 3 standards×8 wells each=total24 wells).

4) Load 100 nanograms (in 1 microliter volume) of one or more samplesindividually into the remaining wells of the plate. Preferably, eachsample is loaded into two or three wells in order to provide replicateassays for each sample. Thus, 36 (in duplicate) or 24 (in triplicate)samples can be assessed per plate.

5) Cover each well with a MICROAMP™ Optical Cap (PE Biosystems partnumber N801-0935).

6) Insert the plates into an ABIPRISM™ 7700 Sequence Detector apparatus.Adjust the settings of the apparatus to select single reporter (FAM),real-time detection.

7) Perform a PCR using the following cycling conditions:

a) 50° C. for 2 minutes.

b) 95° C. for 10 minutes.

c) 40 cycles of 95° C. for 30 seconds and 62° C. for 1 minute. (Totalrun time: 2 hours 6 minutes)

8) Save the data and adjust the setting of the apparatus to select“Allelic Discrimination. ” Select VIC and FAM reporters and TAMRAquencher. Collect data from the plate containing amplified reactionmixtures.

9) Analyze the data using the “Allelic Discrimination” setting of theapparatus. It can be advisable to repeat this protocol for any samplewherein DNA in replicate wells does not clearly fall into one of thethree allelic groups (AA, AG, GG) or wherein DNA in at least one well isnot amplified.

EXAMPLE 3

Allelic Discrimination Assay Using SNPs

Located Upstream of the odc Gene Promoter

There are two single nucleotide polymorphic (SNP) sites that wereidentified upstream of the human odc gene promoter. One SNP is locatedat position −3175 and another SNP is located at position −3004, relativeto the transcriptional start site, of the odc gene. The A-allele of theodc gene comprises a cytosine residue at position −3175, and theG-allele comprises a thymine residue at this position. The A-allelecomprises a guanine residue at position −3004, and the G-allelecomprises a cytosine residue at this position. An allelic discriminationassay was performed on several individual DNA samples using a protocolsimilar to that presented in Example 2, with the followingmodifications.

The following primers and probes were used to analyze the polymorphicnucleotide residue at position −3175.

“forward primer 1”: 5′-TTGAAACTGG GAGGCAGAGG-3′ (SEQ ID NO: 16) “reverseprimer 1”: 5′-CAGAGTTCTG TATAAATCCA CTGACC-3′ (SEQ ID NO: 17)

When used simultaneously in a PCR reaction, forward primer 1 and reverseprimer 1, amplify a 255 base pair fragment including the polymorphicnucleotide residue at −3175.

“TAQMAN™ Allele A probe 1”: 5′-VIC-ATGAAGGAGG TATCTTTTTTCTTCTTTG-TAMRA-3′ (SEQ ID NO: 20) “TAQMAN™ Allele G probe 1”:5′-FAM-ATGAAGGAAG TATCTTTTTT CTTCTTTG-TAMRA-3′ (SEQ ID NO: 21)

The following primers and probes were used to analyze the polymorphicnucleotide residue at position −3004.

“forward primer 2“: 5′-TTTCAGCCAG TCCAACCAC-3′ (SEQ ID NO:18) ”reverseprimer 2”: 5′-ACTCCCATTT TCTTAGGATT CC-3′ (SEQ ID NO:19)

When used simultaneously in a PCR reaction, forward primer 2 and reverseprimer 2 amplify a 143 base pair fragment including the polymorphicnucleotide residue at position −3004.

“TAQMAN™ Allele A probe 2”: 5′-VIC-GACATCACTC TCTCTCTCTGGGTTCCAG-TAMRA-3′ (SEQ ID NO:22) “TAQMAN™ Allele G probe 2”:5′-FAM-GACATCACTC TGTCTCTCTG GGTTCCAG-TAMRA-3′ (SEQ ID NO:23)

Each reaction included 0.1 microgram of genomic DNA, 30 picomoles eachof 2 amplification primers (forward primer 1 and reverse primer 1, orforward primer 2 and reverse primer 2), 12.5 picomoles each of twoTAQMAN™ probes (Allele A probe 1 and Allele G probe 1, or Allele A probe2 and Allele G probe 2) and 1× TAQMAN™ Master Mix reagent (PEBiosystems) in a volume of 50 microliters. PCR cycling conditions were:

a) 50° C. for 2 minutes

b) 95° C. for 10 minutes

c) 40 cycles of 95° C. for 30 seconds and 60° C. for 1 minute. Theresults were analyzed on a PE Biosystems ABI PRISM™ Model 7700 SequenceDetection System using allelic discrimination software supplied by themanufacturer.

The disclosures of every patent, patent application, and publicationcited herein are incorporated herein by reference.

While this invention has been disclosed with reference to specificembodiments, other embodiments and variations of this invention can bedevised by others skilled in the art without departing from the truespirit and scope of the invention. The appended claims include all suchembodiments and equivalent variations.

36 1 59 DNA Homo sapiens 1 gggccccggg cacgtgtgcg gcgcgcctcg ccggcctgcagagacacgtg gtcgccgag 59 2 59 DNA Homo sapiens 2 gggccccggg cacgtgtgcggcgcgcctcg ccggcctgcg gagacacgtg gtcgccgag 59 3 20 DNA ArtificialSequence First pair of primers in Example 1 3 atcgtggctg gtttgagctg 20 424 DNA Artificial Sequence First pair of primers in Example 1 4gtcatctgct ctgtagacac agcg 24 5 22 DNA Artificial Sequence Second(nested) pair of primers in Example 1 5 ggtgctataa gtagggagcg cc 22 6 20DNA Artificial Sequence Second (nested) pair of primers in Example 1 6ccgaagggtt gggaaagagg 20 7 6 DNA Artificial Sequence Hexanucleotide coreof Myc/Max dimer binding site 7 cacgtg 6 8 31 DNA Artificial Sequence Aallele detection probe in Example 1 8 ccggcctgca gagacacgtg gtcgccgagc g31 9 31 DNA Artificial Sequence A allele detection probe in Example 1 9ggccggacgt ctctgtgcac cagcggctcg c 31 10 31 DNA Artificial Sequence Gallele detection probe in Example 1 10 ccggcctgcg gagacacgtg gtcgccgagcg 31 11 31 DNA Artificial Sequence G allele detection probe in Example 111 ggccggacgc ctctgtgcac cagcggctcg c 31 12 17 DNA Artificial SequenceForward primer for allelic discrimination method of Example 2 12cctgggcgct ctgaggt 17 13 17 DNA Artificial Sequence Reverse primer forallelic discrimination method of Example 2 13 aggaagcggc gcctcaa 17 1420 DNA Artificial Sequence TaqMan (TM) allele A probe in Example 2 14cacgtgtctc tgcaggccgg 20 15 19 DNA Artificial Sequence TaqMan (TM)allele G probe in Example 2 15 cacgtgtctc cgcaggccg 19 16 20 DNAArtificial Sequence Forward primer in Example 3 16 ttgaaactgg gaggcagagg20 17 26 DNA Artificial Sequence Reverse primer in Example 3 17cagagttctg tataaatcca ctgacc 26 18 19 DNA Artificial Sequence Forwardprimer in Example 3 18 tttcagccag tccaaccac 19 19 22 DNA ArtificialSequence Reverse primer in Example 3 19 actcccattt tcttaggatt cc 22 2028 DNA Artificial Sequence A allele TaqMan (TM) probe for polymorphicbase at -3176 20 atgaaggagg tatctttttt cttctttg 28 21 28 DNA ArtificialSequence G allele TaqMan (TM) probe for polymorphic base at -3176 21atgaaggaag tatctttttt cttctttg 28 22 28 DNA Artificial Sequence A alleleTaqMan (TM) probe for polymorphic base at -3004 22 gacatcactc tctctctctgggttccag 28 23 28 DNA Artificial Sequence G allele TaqMan (TM probe forpolymorphic base at -3004 23 gacatcactc tgtctctctg ggttccag 28 24 446DNA Homo sapiens 24 ttgaaactgg gaggcagagg ttgcagtgag ccagtattgcgccactgcac tccagcctgg 60 gcaacagagc aagcctcttg actcaaataa taataataataatgataagg gtgactgctt 120 aagtctgcaa tcactatata cctttataag ccttttcatgtaagttagca aagaagaaaa 180 aagatacctc cttcatcaga catgtttttg gttttggtttagaaaacttg gtcagtggat 240 ttatacagaa ctctgtgaaa taagtagatt cttagaacattagaaggaaa gaagaacctg 300 agctttcagc cagtccaacc accccatttt cagaaaggtagtctggaacc cagagagaga 360 gagtgatgtc caaactttca taatgagcgg ggcagagcggcgggggggtg gtaagggggc 420 ccaaggaatc ctaagaaaat gggagt 446 25 446 DNAHomo sapiens 25 ttgaaactgg gaggcagagg ttgcagtgag ccagtattgc gccactgcactccagcctgg 60 gcaacagagc aagcctcttg actcaaataa taataataat aatgataagggtgactgctt 120 aagtctgcaa tcactatata cctttataag ccttttcatg taagttagcaaagaagaaaa 180 aagatacttc cttcatcaga catgtttttg gttttggttt agaaaacttggtcagtggat 240 ttatacagaa ctctgtgaaa taagtagatt cttagaacat tagaaggaaagaagaacctg 300 agctttcagc cagtccaacc accccatttt cagaaaggta gtctggaacccagagagaca 360 gagtgatgtc caaactttca taatgagcgg ggcagagcgg cgggggggtggtaagggggc 420 ccaaggaatc ctaagaaaat gggagt 446 26 234 DNA Homo sapiensmisc_feature (103)..(103) n = c or t 26 ctggctccct gagccaagaa ctggcctctacgttccagtt aatgtttcta tctgaccacc 60 cccaggctgc taggggaacc ctacctgccaagacatccag gcnctctgcc tggaggctgc 120 atactgggca gtgctgggaa cgactggttgataggtgtgc cgggaagctg ggcgattctg 180 gcagtgcagg acacttccgg agtgtgaggaaaattgcgtc attggaacaa agtg 234 27 234 DNA Homo sapiens misc_feature(116)..(116) n = g or t 27 tcaggctccg gcgtctgcgc ttccccatgg ggctggcctgcggcgcctgg gcgctctgag 60 gtgagggact ccccggccgc ggaggaaggg agggagcgagggcgggagcc ggggcnggct 120 gcgggccccg ggccccgggc acgtgtgcgg cgcgcctcgccggcctgcag agacacgtgg 180 tcgccgagcg ggccacgacc ttgaggcgcc gcttcctcccggcccggggt tctc 234 28 234 DNA Homo sapiens misc_feature (155)..(155) n= c or t 28 taaattcctt tttggaatat ttcaaaattt aagtgtctta actaataccacaatgggctg 60 aagtgtcttg gtgtgatatt ttgagtgatt tctttgtgct gtctgacattacacttgata 120 ccatttggtt ttctaaagtg tgaatcagct ttccnagaag tcttggataattggttacat 180 tggaaatcat ggctcacacc tgtaatccag cacttgggga ggccaaggtggtag 234 29 234 DNA Homo sapiens misc_feature (152)..(152) n = g or a 29tttataaaat acatccacat ggtttgttaa aatcatgacg taggcagaat aggattttta 60tcctgttggc atgtatttgt taaaatgttt tgacatcttg atgccttcct aggtagtagt 120tagttgcgta ctgttctttg ataaaaatca tncccataac atcctaaagg agatagggtg 180cctggagggg aatgaaaacg agccacctgg gatatgtagc ctggttttca ggga 234 30 234DNA Homo sapiens misc_feature (154)..(154) n can be t 30 agaggactggtcacaacacg tgtaattaag tagtacttcc tctctccgtc tctttatata 60 gagacctaaaccagatgaga agtattattc atccagcata tggggaccaa catgtgatgg 120 cctcgatcggattgttgagc gctgtgacct gccngaaatg catgtgggtg attggatgct 180 ctttgaaaacatgggcgctt acactgttgc tgctgcctct acgttcaatg gctt 234 31 234 DNA Homosapiens misc_feature (86)..(86) n = c or t 31 tctcatgccc agttaggagtgagtcagggt ttttaatatg ccactttttc tttctcaggc 60 aactcatgca gcaattccagaacccngact tcccacccga agtagaggaa caggatgcca 120 gcaccctgcc tgtgtcttgtgcctgggaga gtgggatgaa acgccacaga gcagcctgtg 180 cttcggctag tattaatgtgtagatagcac tctggtagct gttaactgca agtt 234 32 234 DNA Homo sapiensmisc_feature (95)..(95) n can be t 32 gttttatatg gatttttatt cactcttcagacacgctact caagagtgcc cctcagctgc 60 tgaacaagca tttgtagctt gtacaatggcagaangggcc aaaagcttag tgttgtgacc 120 tgtttttaaa ataaagtatc ttgaaataattaggcattgg gacgttttta tggtgtgttc 180 attccagaca gttcacgaat cccgtatagctcgctctgat tctcagagaa caat 234 33 18 DNA Artificial Sequence Allelicdiscrimination probe for target residue +7487 33 tgggaagtcg gggttctg 1834 18 DNA Artificial Sequence Allelic discrimination probe for targetresidue +7487 34 tgggaagtct gggttctg 18 35 23 DNA Artificial SequenceForward primer for amplification of odc gene region including targetresidue +7487 35 gcaccattgt attccagcct gag 23 36 21 DNA ArtificialSequence Reverse primer for amplification of odc gene region includingtarget residue +7487 36 cgtttcatcc cactctccca g 21

We claim:
 1. A kit for assessing susceptibility of a human to anepithelial cancer, the kit comprising a first oligonucleotide probewhich has a length of about from 20 to 40 nucleotide residues and whichanneals specifically with a target portion of the human's genome,wherein the target portion includes a target nucleotide residue at aposition selected from the group consisting of −3175, −3004, −1936,+263, +5294, +5915, +6697, +7487, and +7886, relative to thetranscription start site, of the odc gene.
 2. The kit of claim 1,wherein the first probe comprises a fluorescent label and a fluorescencequencher attached to separate nucleotide residues thereof.
 3. The kit ofclaim 1, further comprising a first oligonucleotide primer foramplifying a reference portion of the odc gene, the reference portionincluding the target residue.
 4. The kit of claim 3, further comprisinga DNA polymerase having 5′→3′ exonuclease activity.
 5. The kit of claim1, wherein the target residue is at position −3175.
 6. The kit of claim1, wherein the target residue is at position −3004.
 7. The kit of claim1, wherein the target residue is at position −1936.
 8. The kit of claim1, wherein the target residue is at position +263.
 9. The kit of claim1, wherein the target residue is at position +5294.
 10. The kit of claim1, wherein the target residue is at position +5915.
 11. The kit of claim1, wherein the target residue is at position +6697.
 12. The kit of claim1, wherein the target residue is at position +7487.
 13. The kit of claim1, wherein the target residue is at position +7886.
 14. The kit of claim1, wherein the first probe is completely complementary to the targetportion if the target portion corresponds to the A-allele of the odcgene.
 15. The kit of claim 14, further comprising a secondoligonucleotide probe which is completely complementary to the targetportion if the target portion corresponds to the G-allele of the odcgene.
 16. The kit of claim 1, further comprising a secondoligonucleotide probe which has a length of about from 20 to 40nucleotide residues and which anneals specifically with a target portionof the human's genome, wherein the target portion includes a targetnucleotide residue at a position selected from the group consisting of−3175, −3004, −1936, +263, +317, +5294, +5915, +6697, +7487, and +7886,relative to the transcription start site, of the odc gene.
 17. The kitof claim 16, wherein the first and second probes are attached todiscrete regions of a single surface.
 18. The kit of claim 16, whereinthe first and second probes are attached to discrete particles.
 19. Thekit of claim 16, further comprising oligonucleotide primers foramplifying at least the target portion.
 20. The kit of claim 19, whereinthe first and second oligonucleotide probes have the nucleotidesequences SEQ ID NOs: 20 and 21, and the oligonucleotide primers havethe nucleotide sequences SEQ ID NOs: 16 and
 17. 21. The kit of claim 19,wherein the first and second oligonucleotide probes have the nucleotidesequences SEQ ID NOs: 22 and 23, and the oligonucleotide primers havethe nucleotide sequences SEQ ID NOs: 18 and
 19. 22. An isolatedpolynucleotide having a length of from about 15 to 100 nucleotideresidues and being either homologous with or complementary to a portionof the human odc gene, the portion including the nucleotide residue at aposition selected from the group consisting of −3175, −3004, −1936,+263, +5294, +5915, +6697, +7487, and +7886, relative to thetranscription start site, of the odc gene.
 23. The isolatedpolynucleotide of claim 22, having a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 20-23.
 24. An isolatedpolynucleotide having a length of from about 15 to 100 nucleotideresidues and being either homologous with or complementary to a portionof the G-allele of the human odc gene, the portion including one of theguanine residue at position +317, the thymine residue at position −3175,and the cytosine residue at position −3004.
 25. A kit for assessingsusceptibility of a mammal to an epithelial cancer, the kit comprisinga) a first oligonucleotide probe which anneals specifically with atarget portion of the mammal's genome, wherein the target portionincludes a target nucleotide residue at a position selected from thegroup consisting of −3175, −3004, −1936, +263, +5294, +5915, +6697,+7487, and +7886, relative to the transcription start site, of the odcgene, the first probe comprising a fluorescent label and a fluorescencequencher attached to separate nucleotide residues thereof, and b) afirst primer for amplifying a reference portion of the odc gene, thereference portion including the target nucleotide residue.
 26. The kitof claim 15, wherein the first and second oligonucleotide probes havethe nucleotide sequences TGGGAAGTCG GGGTTCTG (SEQ ID NO: 33) andTGGGAAGTCT GGGTTCTG (SEQ ID NO: 34).
 27. The kit of claim 19, whereinthe first and second oligonucleotide probes have the nucleotidesequences TGGGAAGTCG GGGTTCTG (SEQ ID NO:33) and TGGGAAGTCT GGGTTCTG(SEQ ID NO:34), and the oligonucleotide primers have the nucleotidesequences GCACCATTGT ATTCCAGCCT GAG (SEQ ID NO:35) and CGTTTCATCCCACTCTCCCA G (SEQ ID NO:36).
 28. The isolated polynucleotide of claim22, having a nucleotide sequence selected from the group consisting ofTGGGAAGTCG GGGTTCTG (SEQ ID NO:33) and TGGGAAGTCT GGGTTCTG (SEQ IDNO:34).
 29. The isolated polynucleotide of claim 22, having a length offrom about 20 to 40 nucleotide residues.
 30. The isolated polynucleotideof claim 22, having a length of from about 20 to 30 nucleotide residues.